The present invention relates to the field of lactic acid bacterial starter cultures and in particular there is provided the means of metabolically engineering such bacteria to obtain mutants or variants hereof which, when they are used in the manufacturing of fermented food products produce increased amounts of desirable metabolites or reduced amounts of less desirable metabolites.
Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus species including Lactococcus lactis are among the most commonly used lactic acid bacteria in dairy starter cultures. However, several other lactic acid bacteria such as Leuconostoc species, Pediococcus species, Lactobacillus species and Streptococcus species. Species of Bifidobacteri-um, a group of strict anaerobic bacteria, are also commonly used in food starter cultures alone or in combination with lactic acid bacterial species.
When a lactic acid bacterial starter culture is added to milk or any other food product starting material under appropriate conditions, the bacteria grow rapidly with concomitant conversion of citrate, lactose or other sugar compounds into lactic acid/lactate and possibly other acids including acetate, resulting in a pH decrease. In addition, several other metabolites are produced during the growth of lactic acid bacteria. These metabolites include ethanol, formate, acetaldehyde, xcex1-acetolactate, acetoin, diacetyl, and 2,3 butylene glycol (butanediol). Among these metabolites, diacetyl is an essential flavour compound which is formed during fermentation of the citrate-utilizing species of e.g. Lactococcus, Leuconostoc and Lactobacillus. Diacetyl is formed by an oxidative decarboxylation (FIG. 1) of xcex1-acetolactate which is formed by the action of xcex1-acetolactate synthetase (Als) from two molecules of pyruvate. Pyruvate is a key intermediate of several lactic acid bacterial metabolic pathways including the citrate metabolism and the degradation of lactose or glucose to lactate. The pool of pyruvate in the cells is critical for the flux through the metabolic pathway leading to diacetyl, acetoin and 2,3 butylene glycol (butanediol) via the intermediate compound xcex1-acetolactate due to the low affinity of xcex1-acetolactate synthetase for pyruvate.
Pyruvate is converted to formate and acetyl coenzyme A (acetyl CoA) (FIG. 1) by the action of pyruvate formate-lyase (Pfl). This conversion takes place only under anaerobic conditions (Frey et al. 1994). Pfl is inactivated even at low levels of oxygen, and a switch from anaerobic to aerobic conditions will lead to significant changes in metabolic end product profiles in lactic acid bacteria with complete disappearance of ethanol and formate (Hugenholtz, 1993). Another factor which regulates the activity of Pfl is the pH. The pH optimum of Pfl is about 7 (Hugenholtz, 1993).
An alternative pathway for the formation of acetyl CoA from pyruvate (FIG. 1) in a lactic acid bacterium is by the activity of the pyruvate dehydrogenase complex (PDC). In contrast to Pfl, PDC has a very low activity under anaerobic conditions due to the inhibitory effect of NADH on that enzyme (Snoep et al. 1992). This enzyme requires the presence of lipoic acid as a co-factor to be active.
Additionally, acetyl CoA can be produced in lactic acid bacteria from acetate under aerobic as well as under anaerobic conditions.
Accordingly, it is conceivable that the pyruvate pool is increased under anaerobic conditions if the lactic acid bacterial strain is defective in enzyme systems involved in pyruvate consumption, including Pfl. As mentioned above, an increased pyruvate pool may lead to an increased flux from pyruvate towards acetoin and diacetyl or other metabolites derived from xcex1-acetolactate. Thus, it is to be expected that fermented food products which are produced by using a lactic acid bacterial starter culture having a reduced Pfl activity or completely lacking such activity contain an increased amount of acetoin or other of the above metabolites. Conversely, the such starter cultures may produce reduced amounts of other metabolites, including ethanol and acetate and possibly, acetaldehyde.
Recent studies have shown that when L. lactis is lacking the lactate dehydrogenase (Ldh) which is involved in the major pyruvate consuming pathway leading to lactate, more pyruvate is directed towards acetoin and butanediol via xcex1-acetolactate, possibly resulting in increased formation of the intermediate product diacetyl (Platteeuw et al., 1995; Gasson et al., 1996).
Overproduction of xcex1-acetolactate synthetase in Lactococcus lactis as another approach of metabolically engineering lactic acid bacteria to produce increased amounts of diacetyl has been disclosed by Platteeuw et al. 1995.
The potential of using L. lactis strains with reduced pyruvate formate-lyase activity as a means of increasing diacetyl formation is mentioned by Hugenholtz, 1993. It is suggested by this author that the combination of three strategies: 1) Ldh inactivation by mutation/genetic engineering, 2) Pfl inactivation by aeration and/or low pH and 3) acetolactate decarboxylase (ALD) inactivation by mutation/genetic engineering could result in a high production of xcex1-acetolactate from lactose.
However, the suggested inactivation of Pfl activity by aeration and/or low pH is not feasible or possible in the industrial production of lactic acid bacterially fermented dairy products or other fermented food products, as the production hereof generally takes place under essentially anaerobic conditions. Furthermore, the pH of the starting materials including milk is typically about 7 and it is generally not desirable to lower the pH of the food material to be fermented.
Whereas it has been suggested to modify the Pfl activity of lactic acid bacteria as a means of changing their production of metabolites in a desirable direction by manipulating the growth conditions, there have been no suggestions in the prior art to utilize metabolically engineered lactic acid bacteria which have a modified Pfl activity under industrially appropriate and feasible culturing conditions.
A method that allows isolation of mutants of gram-negative bacteria devoid of Pfl activity has been disclosed by Pascal et al., 1974. This method includes the selection of Pfl defective mutants of E. coli and Salmonella typhimurium based on their lack of ability to generate H2 and CO2 in the absence of formate, when they are incubated under anaerobic condition in media containing glucose or pyruvate. However, such a selection method cannot be used for selection of Pfl defective mutants of lactic acid bacteria, since these organisms lack the enzyme that catalyses production of H2 and CO2 from formate.
Accordingly, the prior art does not contain any guidance with respect to designing a feasible method of isolating a lactic acid bacterial Pfl defective (Pflxe2x88x92) mutant.
Experiments performed by the inventor with the minimal medium BA (Clark and Maalxc3x8e, 1967) for E. coli, showed that this medium did not support the aerobic growth of lactic acid bacteria. However, if cultivated in this medium together with E. coli the growth of lactic acid bacteria was supported, indicating that E. coli produces a factor needed for the growth of the lactic acid bacteria. It has later been found that this growth factor is acetate, which led to the development of the DN-medium (Dickely et al., 1995).
It has now surprisingly been found that wild-type strains of lactic acid bacteria such as strains of Lactococcus and Streptococcus including as examples Lactococcus lactis and Streptococcus thermophilus strains under anaerobic conditions grow well on the DN-medium (Dickely et al., 1995) in the absence of acetate. These unexpected findings have made it possible to develop a novel and simple method for the isolation of Pfl defective lactic acid bacterial mutants based on the finding that such mutants, in contrast to the phenotypically Pfl+ wild-type strains, are unable to grow under anaerobic conditions on DN-medium in the absence of acetate.
Additionally, having such a method allowing the selection of Pfl defective lactic acid bacterial mutants at hand has made it possible to provide further mutated cells which in addition to being Pflxe2x88x92 are mutated in one or more genes involved in the citrate/sugar metabolic pathways such as e.g. the ldh gene coding for lactate dehydrogenase (Ldh) so as to provide a variety of metabolically engineered lactic acid bacteria having highly desirable improved characteristics with respect to metabolite (fermentation end product) production.
The above findings have thus opened up for a novel approach for providing useful metabolically engineered lactic acid bacterial starter cultures which approach is based on relatively simple classical random mutagenesis methods or the selection of spontaneously occurring mutants and which does not involve in vitro genetic engineering. From a practical technological point of view this is advantageous, since in most countries the use of genetically engineered food starter cultures is still conditional on approval by regulatory bodies.
Accordingly, the invention provides in a first aspect a method of isolating a pyruvate formate-lyase (Pfl) defective lactic acid bacterium, the method comprising the steps of
(i) providing a wild-type lactic acid bacterial strain which under aerobic conditions is not capable of growth in the absence of acetate in a medium not containing lipoic acid, but which is capable of growth is such medium under anaerobic conditions, and
(ii) selecting from said wild-type strain a mutant which under said conditions essentially does not grow in the absence of acetate.
In a further aspect, the invention relates to a Pfl defective mutant lactic acid bacterium which is obtainable by the above method and having, relative to the wild-type strain from which it is derived, at least one of the following characteristics:
(i) essentially the same growth rate when cultivated under aerobic conditions in M17 medium,
(ii) a reduced growth rate or a reduced rate of acid production when cultivated under anaerobic conditions in M17 medium or in reconstituted skim milk (RSM),
(iii) essentially no production of formate under the anaerobic conditions of (ii),
(iv) a reduced production of ethanol or acetate under said above anaerobic conditions, and/or
(vi) an increased production of at least one xcex1-acetolactate-derived metabolite when cultivated under anaerobic conditions in RSM.
In a still further aspect, there is provided a method of isolating a Pfl and lactate dehydrogenase (Ldh) defective lactic acid bacterium which is not capable of growth under anaerobic conditions in the presence of acetate, said method comprising
initially selecting a Pfl defective lactic acid bacterium in accordance with the above method, and
(ii) selecting from said Pfl defective lactic acid bacterium a strain which is incapable of growing under anaerobic condition in an acetate-containing medium.
The invention pertains in another aspect to a Pfl and Ldh defective mutant lactic acid bacterium which is not capable of growing under anaerobic conditions in the presence of acetate, said bacterium being obtainable by the above method of isolating a Pfl and lactate dehydrogenase (Ldh) defective lactic acid bacterium, and having, relative to a wild-type lactic acid bacterium or its Pfl defective parent strain, at least one of the following characteristics:
(i) essentially the same growth yield when cultivated under aerobic conditions in M17 medium,
(ii) a reduced capability of converting lactose to lactate,
(iii) an increased production of xcex1-acetolactate, and/or
(iv) an increased production of an xcex1-acetolactate derived metabolite.
In further aspects, the invention relates to a mutant or variant of the above Pfl and Ldh defective mutant which mutant or variant is capable of growing anaerobically, to a method of producing a food product, comprising adding to the food product starting materials a culture of any of the above mentioned lactic acid bacteria and a method of producing a lactic acid bacterial metabolite, comprising cultivating any of the above mentioned lactic acid bacteria under conditions where the metabolite is produced, and isolating the metabolite from the culture.
There is also provided a lactic acid bacterial starter culture composition comprising any of the above mentioned lactic acid bacteria.
The present invention provides in a first aspect a method of isolating a pyruvate formate-lyase (Pfl) defective mutant lactic acid bacterium. As used herein the expression xe2x80x9cpyruvate formate-lyase defectivexe2x80x9d indicates that the lactic acid bacterial mutant as compared to the wild-type parent strain has a reduced Pfl activity or that the Pfl activity is absent irrespective of the growth conditions, Plf activity being expressed herein in terms of formate production. Such a mutant strain is also referred to herein as a strain having a Pflxe2x88x92 phenotype.
As used herein, the expression xe2x80x9clactic acid bacteriumxe2x80x9d designates gram positive, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Pediococcus species and Brevibacterium species. Also the strict anaerobes belonging to the genus Bifidobacterium is generally included in the group of lactic acid bacteria.
A lactic acid bacterial mutant as defined above can be derived by selecting a spontaneously occurring mutant of a wild-type strain of a lactic acid bacterium which has the characteristic that it, when it is cultivated under aerobic conditions in a medium which does not contain lipoic acid, has a growth requirement for acetate, but which under anaerobic conditions is capable of growing in such a medium in the absence of acetate. Alternatively, the mutant of the wild-type lactic acid bacterial strain can be provided by subjecting the strain to a mutagenization treatment prior to the selection of a mutant having the above characteristics of the Pfl defective strain.
It is assumed that these different requirements for acetate under the above aerobic and anaerobic conditions, respectively is caused by the facts that under aerobic conditions insufficient amounts of acetyl CoA is formed by the lactic acid bacterium due to at least two circumstances: (i) in the absence of lipoic acid, an essential co-factor for the activity of the acetyl CoA generating pyruvate dehydrogenase complex (PDC), this enzyme complex does not generate acetyl CoA and (ii) the other major acetyl CoA generating enzyme, pyruvate formate-lyase (Pfl) is inactivated in the presence of oxygen. Therefore, under such aerobic conditions, the wild-type lactic acid bacterium requires acetate as an alternative source of acetyl CoA. In contrast, under anaerobic conditions, the Pfl is activated and assumingly provides acetyl CoA in sufficient amounts for growth of the bacterium. As it is mentioned above, these observations were the starting point for designing the present method of isolating a Pfl defective mutant of a lactic acid bacterium as described herein and the use hereof as an intermediate for providing further modified strains of lactic acid bacteria.
In accordance with one embodiment of the invention, this method provides in a first step the provision of a wild-type lactic acid bacterium having the above acetate requirement characteristics, followed by subjecting the bacterium to a mutagenization treatment. In accordance with the invention, suitable mutagens include conventional chemical mutagens and UV light. Thus, as examples, a chemical mutagen can be selected from (i) a mutagen that associates with or become incorporated into DNA such as a base analogue, e.g. 2-amino-purine or an interchelating agent such as ICR-191, (ii) a mutagen that react with the DNA including alkylating agents such as nitrosoguanidine or hydroxylamine, or ethane methyl sulphonate (EMS).
Although the lactic acid bacterial mutant can be provided by subjecting a parent strain to a chemical mutagenization treatment followed by selecting a Pflxe2x88x92 mutant, it will be understood that it would also be possible to provide the mutant by selecting a spontaneously occurring mutant in accordance with the selection procedure as described herein. As an alternative to one presently preferred method of providing the mutant by random mutagenesis, it is also possible to provide such a mutant by site-directed mutagenesis, e.g. by using appropriately designed PCR techniques or by using a transposable element which is integratable in lactic acid bacterial replicons.
When a mutagenization step is included, the mutagenized strain is, subsequent to the mutagenization treatment, cultivated under anaerobic conditions in a defined medium not containing lipoic acid in the absence or presence, respectively of acetate, and a mutant strain, which in contrast to the wild-type parent strain essentially does not grow under these conditions in the absence of acetate, is selected. It is assumed that such a mutant strain has a defect in the gene coding for the Pfl polypeptide implying that the production of the enzyme is at least partially blocked or that the enzyme is produced in an at least partially inactive form. This assumption can be affirmed by testing the selected mutant for lack of production of formate or alternatively, a reduced pyruvate formate-lyase activity.
When the mutant is provided as a spontaneously occurring mutant the above wild-type strain is subjected to the selection step without any preceding mutagenization treatment. The lactic acid bacterial wild-type parent strain can be selected from any industrially suitable lactic acid bacterial species, i.e. the strain can be selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Leuconostoc species, a Pediococcus species, a Streptococcus species and a Bifidobacterium species. In particular useful embodiments, the lactic acid bacterium is a Lactococcus lactis or a Streptococcus thermophilus. Examples of presently preferred lactic acid bacteria are Lactococcus lactis subspecies lactis and Lactococcus lactis subspecies lactis biovar diacetylactis.
A Pfl defective (Pflxe2x88x92 phenotype) mutant lactic acid bacterium which can be obtained by the above method has, relative to the wild-type parent strain one or more phenotypically recognizable characteristics distinguishing it from the parent strain. Thus, the Pflxe2x88x92 mutant strain may have essentially the same growth rate when cultivated under aerobic conditions in M17 medium but a reduced growth rate or a reduced acid production when cultivated under anaerobic conditions in conventional media such as the M17 medium or reconstituted skim milk (RSM), essentially no production of formate, a reduced production of ethanol or acetate under said above anaerobic conditions and/or an increased production of at least one xcex1-acetolactate-derived metabolite when cultivated under anaerobic conditions, e.g. in RSM.
Several of these characteristics may be desirable for specific purposes. In the production of a food product it may thus be advantageous that the strain produces lesser amounts of acids, formate, acetate or ethanol, whereas an enhanced production of xcex1-acetolactate derived aroma or flavour compounds can be highly desirable, in particular in the production of dairy products. Such desirable compounds include acetoin, diacetyl and 2,3 butylene glycol. In useful embodiments, the production of such metabolites such as acetoin is increased by at least 50%, more preferably by at least 100% and in particular by at least 200%. Besides being useful in the manufacturing of a food product, a mutant strain overproducing xcex1-acetolactate derived metabolites can also be used,in the production of the metabolites as such.
In accordance with the invention, a Pfl defective (Pflxe2x88x92) mutant strain is selected from a Lactococcus species, a Lactobacillus species, a Leuconostoc species, a Pediococcus species, a Streptococcus species and a Bifidobacterium species. In this context, one preferred species is Lactococcus lactis including Lactococcus lactis subspecies lactis and Lactococcus lactis subspecies lactis biovar diacetylactis, e.g. the Lactococcus lactis subspecies lactis strain DN221 which has been deposited under the accession No. DSM 11034, or a Lactococcus lactis strain having essentially the characteristics of that strain, or the Lactococcus lactis subspecies lactis biovar diacetylactis strain DN227 which has been deposited under the accession No. 11040, or a Lactococcus lactis strain having essentially the characteristics of that strain.
It will be understood that the Plf defective lactic acid bacterial mutant can be utilized as a host for the cloning of a pfl gene by complementation of the defective gene. Importantly, the Plfxe2x88x92 strain can also be used as a parent strain for isolating mutants having further useful enzymatic defects as it will be described in the following.
Lactate dehydrogenase (Ldh) is, as it can be seen from FIG. 1, another enzyme which in lactic acid bacteria contribute to the consumption of the pyruvate pool, the activity of the enzyme predominantly resulting in the production of lactate. It was contemplated that the metabolic flux towards xcex1-acetolactate and metabolites derived from this intermediate could be further increased by providing a mutant strain which in addition to having a defect in the Pfl activity is defective in Ldh.
Therefore, a strategy for isolating and selecting a lactic acid bacterium which in addition to being Pfl defective is also Ldh defective (Ldhxe2x88x92), i.e. having the Pflxe2x88x92 Ldhxe2x88x92 phenotypes, was developed based on the following considerations: During anaerobic growth of wild-type lactic acid bacteria the NADH being produced in the glycolysis is converted to NAD+ during production of lactate and to some extent during the production of ethanol. Accordingly, it was hypothesized that a double mutant having the Pflxe2x88x92 Ldhxe2x88x92 phenotype would be unable to grow under anaerobic conditions, i.e. such a strain would have the additional phenotype Angxe2x88x92 (inability to grow anaerobically). This hypothesis was based on the assumption that such a double mutant would be unable to regenerate NAD+ from NADH under anaerobic conditions, since Pfl would be blocked by a mutation (whereas under aerobic conditions, NADH can be converted to NAD+ by NADH oxidase), PDC would be blocked due to inhibition by NADH and Ldh would be blocked by mutation. It was thus contemplated that a Pflxe2x88x92 Ldhxe2x88x92 double mutant could grow under aerobic conditions but not under anaerobic conditions.
Based on the above considerations, a method of isolating a Pfl and lactate dehydrogenase (Ldh) defective lactic acid bacterium which is not capable of growth under anaerobic conditions in the presence of acetate, i.e. a Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 phenotype, was developed. The method comprises as a first step, the selection of a Pfl defective lactic acid bacterium in accordance with the above method, followed by selecting from this Pfl defective bacterium a strain which is incapable of growing under anaerobic conditions in an acetate-containing medium.
In one presently preferred embodiment this method includes the step of subjecting, prior to selection of a strain which is incapable of growing under anaerobic conditions in an acetate-containing medium, the Pfl defective lactic acid bacterium to a mutagenization treatment and subsequently selecting a mutant which under said conditions essentially does not grow under said anaerobic conditions.
The above method of isolating the Plf and Ldh defective mutant results in a strain having an Ldh specific activity which is reduced relative to that of its parent (Pfl defective) strain. Preferably, the thus selected mutant has an Ldh specific activity which is less than 10 units/mg protein of a cell free extract of the bacterium.
Typically, the thus reduced Ldh specific activity corresponds to at the most 50% activity relative to the wild-type or Pflxe2x88x92 parent strain, such as at the most 25% or preferably, at the most 10% activity such as at the most 5% relative to the parent strains. It is particularly preferred that the mutant strain essentially is devoid of Ldh activity.
The mutagenization step whereby the Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant is produced from the Pflxe2x88x92 mutant can be performed according to the methods as described above for the mutagenization of the wild-type strain. It follows from the above description of this initial step of providing the Pflxe2x88x92 mutant that useful strains can be selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Leuconostoc species, a Pediococcus species, a Streptococcus species and a Bifidobacterium species. A presently preferred lactic acid bacterium is Lactococcus lactis including Lactococcus lactis subspecies lactis and Lactococcus lactis subspecies lactis biovar diacetylactis.
In accordance with the invention there is also provided a Pfl and Ldh defective mutant lactic acid bacterium which is obtainable by the above method. In addition to its Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 phenotypes, such a mutant strain can be distinguished from a wild-type lactic acid bacterium or its Pfl defective parent strain in one or more further characteristics. Thus, the mutant strain may have essentially the same growth yield when cultivated under aerobic conditions in M17 medium, a reduced capability of converting lactose to lactic acid/lactate, increased production of xcex1-acetolactate and/or an increased production of an xcex1-acetolactate derived metabolite. Surprisingly, the production of xcex1-acetolactate and/or metabolites derived from xcex1-acetolactate was not only increased under aerobic conditions where the mutant strain can grow, but also under anaerobic conditions where essentially no growth occurred.
As it is shown in the below Examples, the increase of production of xcex1-acetolactate and metabolites derived therefrom was of a significant magnitude. Thus, Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutants according to the invention preferably have a production of xcex1-acetolactate and/or metabolites derived therefrom which, relative to a wild-type strain of the same species, is increased by at least 50%, such as by at least 100%. It is even more preferred the production is increased by at least 200% such as at least 1000%.
In accordance with the invention, the Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant can be of any lactic acid bacterial species selected from a Lactococcus species, a Lactobacillus species, a Leuconostoc species, a Pediococcus species, a Streptococcus species and a Bifidobacterium species. One preferred species is Lactococcus lactis including Lactococcus lactis subspecies lactis such as the strain designated DN223 which is described in the following and which is deposited under the accession No. DSM 11036 or a Lactococcus lactis strain having essentially the characteristics of that strain, and Lactococcus lactis subspecies lactis biovar diacetylactis.
As a result of the enzyme defects of the present Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 lactic acid bacterial mutant, such a mutant is capable of converting a substantial proportion of the intracellular pyruvate pool to xcex1-acetolactate and further to one or more of the metabolites which can be formed from this intermediate compound, including acetoin, butanediol and/or diacetyl which latter compound can be formed by chemically oxidizing xcex1-acetolactate. Thus, in one preferred embodiment, the Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant is capable of converting at least 15% of pyruvate being catabolized to acetoin, more preferably at least 30%. In even more preferred embodiments, this conversion is at least 40%, such as at least 50% or even at least 60%.
In a further aspect, the invention relates to a mutant or variant of the above Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant lactic acid bacterium which is capable of growing anaerobically. Such a mutant or variant strain can be provided by selecting a spontaneous mutant of the above mutant bacterium, which mutant or variant strain can grow anaerobically. Alternatively, the mutant or variant strain can be made by subjecting the Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant to a further mutagenization treatment in accordance with a method as described above, and selecting a strain being capable of growing anaerobically. It is contemplated that such mutants or variants would have regained the ability to convert NADH to NAD+ under anaerobic conditions, either by mutations in systems secondary to Ldh or Pfl, or by reversion of the Pflxe2x88x92 phenotype to Pfl+ phenotype. In wild-type lactic acid bacteria, the level of NADH is high and it can be oxidized via lactate and/or ethanol production, i.e. via the pyruvate metabolism. The implication hereof is that lactic acid bacteria produce relatively high levels of lactate and/or ethanol as compared to aerobic conditions. From this it also follows that the metabolites having an aroma effect (diacetyl, acetoin) are only produced at relatively low levels.
It was found that this general picture was still found in the present Ang+ mutant or variant of the Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant. However, it was surprisingly found that such a mutant/variant has, relative to its parent strain and to the wild-type strain, a significantly altered production of aroma compounds under anaerobic growth conditions. Thus, the above Ang+ mutant/variant may be one which has a production of acetaldehyde which relative to the original wild-type strain is increased at least 2-fold, such as at least 5-fold or even at least 8-fold. The mutant variant may also have a production of the diacetyl precursor xcex1-acetolactate which, also relative to the wild-type strain is increased at least 5-fold such as at least 10-fold.
Also, the production of acetoin and/or formate may be significantly increased in such a mutant/variant. Thus, as one typical example, the mutant/variant is one which, when grown anaerobically in reconstituted skim milk powder, produces in excess of 1 mM acetoin and/or in excess of 10 mM formate.
A mutant or variant having the latter characteristic is assumingly Ldh defective but has the wild-type Pfl activity, i.e. it has the phenotype Pfl+ Ldhxe2x88x92 Ang+. One example of such a strain is the Lactococcus lactis subspecies lactis DN224 deposited under the accession No. DSM 11037 or a Lactococcus lactis strain having essentially the characteristics of that strain. Another example of the present mutant or variant is a strain which is Pfl defective and has the wild-type Ldh activity, i.e. having the phenotype Pflxe2x88x92 Ldh+ Ang+.
In addition to being a starting material for providing further lactic acid bacterial mutants or variants, the above Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant can be utilized as host for cloning of genes which can restore the ability of the mutant to grow under anaerobic conditions.
Such a mutant can also, as it is described above by way of example, be used for selecting further mutants having regained the capability of growing anaerobically e.g. due to 30 mutations whereby an increased amount of one or more NADH oxidoreductases is produced. Such oxidoreductases include diacetyl reductase (Dr) and Ldh.
The mutant can also be one in which the mutation results in overproduction and/or enhanced activity of an enzyme, the activity of which can be limiting for a pathway in which a NADH dependent oxidoreductase is involved. Such an overproduction or enhanced activity can e.g. be of the xcex1-acetolactate synthetase (Als), the increased production or activity of which would in turn result in an increased production of substrate for diacetyl reductase. Alternatively, the mutation may result in the above enzyme having an increased activity.
NADH dependent oxidoreductases require a substrate. Thus, as an example, acetoin is the substrate for the oxidoreductase diacetyl reductase (see FIG. 1). Accordingly, it is contemplated that the above Pflxe2x88x92 Ldhxe2x88x92 Angxe2x88x92 mutant can be used for selecting a mutant which does not grow, even if the oxidoreductase substrate such as acetoin is added to the medium. Such a mutant assumingly will have a defect in one or more of its oxidoreductases e.g. diacetyl reductase.
Any of the above mutants or variants are potentially useful in the production of food products and accordingly, the invention relates in a further aspect to a method of producing a food product which method comprises that a culture of a lactic acid bacterium as described herein is added to the food product starting materials which are then kept under conditions appropriate for the bacteria to grow and/or to be metabolically active. The purpose of the addition of the lactic acid bacteria depends of the food product. In some instances, a lactic acid bacterium according to invention is used to provide an increased production in the food product, such as e.g. a dairy product, of a particularly desirable aroma compound, such as diacetyl, acetoin or acetaldehyde. Other examples of food products where use of the present mutant strains is contemplated include meat products, vegetables, bakery products and wine.
It will also be understood that the presently provided strains will be highly useful as production strains in the manufacturing of lactic acid bacterial metabolite compounds including the above aroma compounds. Accordingly, the invention encompasses in a still further aspect a method of producing a lactic acid bacterial metabolite. Such a method comprises cultivating one or more of the lactic acid bacteria as disclosed herein in a suitable medium under industrially feasible conditions where the metabolite is produced, and isolating, if required, the metabolite from the culture. The metabolite can be isolated in accordance with any suitable conventional method of isolating the particular compound(s) from the cultivation medium. It is also possible to use the cultivation medium containing the outgrown culture of lactic acid bacteria directly as a source of one or more metabolites.
A specific example of such a production method for a lactic acid bacterial metabolite is a method of producing what is normally referred to in the art as xe2x80x9cstarter distillatexe2x80x9d which is a diacetyl-containing flavouring product conventionally made by cultivating a conventional wild-type starter culture strain of a lactic acid bacterium which produces acetoin and/or diacetyl in a suitable.medium and isolating the metabolites by distillation to provide a concentrate of the metabolites. This product is used for flavouring of butter, margarine, spreads, cereal products and pop-corn. It has been found that by using the strains DN223 or DN224, such a starter distillate can be obtained that has a content of diacetyl which, in comparison with a conventional starter distillate, is at least 2-fold.
It is convenient to provide the lactic acid bacterium according to the invention, both when it is used as a food production strain and as a production strain for metabolites, as a lactic acid bacterial starter culture composition comprising the lactic acid bacterium selected for the specific use. Typically, such compositions contain the bacterium in concentrated form e.g. at a concentration of viable cells (colony forming units, CFUS) which is in the range of 105 to 1013 per g of the composition such as a range of 106 to 1012 per g. Additionally, the starter culture composition may contain further components such as bacterial nutrients, cryoprotectants or other substances enhancing the viability of the bacterial active ingredient during storage. The composition can be in the form of a frozen or freeze-dried composition.
The invention is further illustrated in the following examples and the drawings wherein.